骨组织工程中细胞因子基因修饰种子细胞来源研究:转染后骨形态发生蛋白-3成纤维细胞株的稳定表达(英文)

Study on the resource of cytokine gene modified seed cells in bone tissue engi neering:the stable expression of fibroblasts after bone morphogenetic protein 3 transfection

背景:骨形态发生蛋白(bonemorphogeneticprotein,BMP)是诱导和促进种子细胞向骨细胞方向转化的最重要的细胞因子之一。天然BMP难溶解于培养基,对培养的种子细胞无法有效发挥作用;可溶性重组BMP虽可溶解于培养基,但价格昂贵,而且难于适时、适量的作用于种子细胞。基因治疗为上述问题的解决提供了新的思路。目的:转染外源BMP-3基因入成纤维细胞,筛选获得稳定表达BMP-3的阳性成纤维细胞克隆。设计:单一样本研究。单位:解放军第四军医大学西京医院全军骨科研究所。材料:成纤维细胞(NIH3T3细胞)由第四军医大学口腔医学院司徒镇强教授提供。方法:2000-04/2003-11在全军重点实验室第四军医大学全军骨科研究所实验室完成。采用脂质体转染的方法将外源BMP-3基因转染入NIH3T3细胞,G418筛选后寻找细胞集落,分离、培养,免疫组织化学鉴定,BMP-3染色阳性者为阳性BMP-3表达细胞克隆。主要观察指标:①NIH3T3细胞筛选浓度。②转染阳性细胞克隆的筛选。③筛选的细胞克隆内BMP-3的表达。结果:成功将BMP-3基因转染入NIH3T3细胞,经免疫组化筛选出阳性BMP-3表达成纤维细胞的克隆,建立了BMP-3稳定表达成纤维细胞株。结论:将BMP-3基因真核表达载体转染入成纤维细胞,并经筛选获得了可稳定表达BMP-3的成纤维细胞株,为将来骨组织工?

BACKGROUND:Bone morphogenetic protein(BMP) is one of the most important cytoki nes that induce and promote seed cells to be transformed into osteocytes.Insolub le natural BMP can hardly affect the life of cultured seed cells.The expensive s oluble recombinant BMP is also hard to work on the seed cells at the appropriate time and dose.Therefore,gene therapy technique provides us with a brand new ide a of using gene modified seed cells. OBJECTIVE:To transfect exogenous BMP 3 gene into the fibroblasts and screen t he positive fibroblast clones that can express BMP 3 stably. DESIGN:Simple sample study. SETTING:Orthopaedic Research Institute,Xijing Hospital,Fourth Military Medical University of Chinese PLA. MATERIALS:The fibroblasts(NIH3T3) were kindly presented by Professor Situ Zhen qiang of the Stomatological College of Fourth Military Medical University of C hinese PLA. METHODS:This experiment was conducted in the Key Laboratory of Chinese PLA,whi ch belongs to the Orthopaedic Research Institute of Fourth Military Medical Univ ersity.BMP 3 gene was transfected into the fibroblasts through lipofectamin.The transfected cells were screened by G418.The separated cloned cells were identif ied through immunohistochemistry.The positively stained cells were the clones of BMP 3 expressing fibroblasts. MAIN OUTCOME MEASURES:The screening concentration of NIH3T3 cells, screening o f positive transfected cells,and expression of BMP 3 in screened cells. RESULTS:BMP 3 gene was successfully transfected into the fibroblasts.BMP 3 e xpressing fibroblast clones were screened and identified through immunohistochem istry.Fibroblast strains with stable BMP 3 expression were obtained. CONCLUSION:The transfection of BMP 3 gene eukaryonic expression vector into t he fibroblasts and obtaining of fibroblast strains with BMP 3 expression have l aid foundation for the usage of gene modified seed cells in future research of bone tissue engineering.